VIP conditions human endometrial receptivity by privileging endoplasmic reticulum stress through ATF6α pathway.

Endometrial stromal cells undergo endoplasmic reticulum (ER) stress and unfolded protein response (UPR) during the decidualization linked with the inflammation and angiogenesis processes. Considering VIP (vasoactive intestinal peptide) induces the decidualization program, we studied whether modulates the ER/UPR pathways to condition both processes for embryo implantation. When Human Endometrial Stromal Cell line (HESC) were decidualized by VIP we observed an increased expression of ATF6α, an ER stress-sensor, and UPR markers, associated with an increase in IL-1ß production. Moreover, AEBSF (ATF6α -inhibitor pathway) prevented this effect and decreased the expansion index in the in vitro model of implantation. VIP-decidualized cells also favor angiogenesis accompanied by a strong downregulation in thrombospondin-1. Finally, ATF6α, VIP and VPAC2-receptor expression were reduced in endometrial biopsies from women with recurrent implantation failures in comparison with fertile. In conclusion, VIP privileged ATF6α-pathway associated with a sterile inflammatory response and angiogenesis that might condition endometrial receptivity.

Impact of the Reticular Stress and Unfolded Protein Response on the inflammatory response in endometrial stromal cells.

During decidualization, endometrial stromal cells undergo reticular stress (RS) and unfolded protein response (UPR), allowing the endoplasmic reticulum-expansion and immunomodulators production. Physiological RS generates the activation of sensing proteins, inflammasome activation and mature-IL-1ß secretion, associated with pro-implantatory effects. We focus on the impact of RS and UPR on decidualized cells and whether they induce a physiological sterile inflammatory response through IL-1ß production. Human endometrial stromal cell line (HESC) after decidualization treatment with MPA + dibutyryl-cAMP (Dec) increased the expression of RS-sensors (ATF6, PERK and IRE1α) and UPR markers (sXBP1 and CHOP) in comparison with Non-dec cells. Then we found increased NLRP3 expression in Dec cells compared with Non-dec cells. In fact STF-083010 (an IRE1α inhibitor) prevented this increase. Downstream, increased levels of active caspase-1 on Dec cells were detected by FAM-Flica Caspase-1 associated with an increase in IL-1ß production. Moreover, the treatment with STF-083010 decreased the invasion index observed in Dec cells, evaluated by an in vitro model of implantation. In endometrial biopsies from recurrent spontaneous abortion patients an increased expression of IRE1α was found in comparison with fertile women; while recurrent implantation failure samples showed a lower expression of sXBP1, TXNIP and NLRP3 than fertile women, suggesting that RS/UPR tenors might condition endometrial receptivity.

Acetylcholine contributes to control the physiological inflammatory response during the peri-implantation period.

BACKGROUND: Maternal antigen-presenting cells attracted to the pregnant uterus interact with trophoblast cells and modulate their functional profile to favour immunosuppressant responses. Non-neuronal cholinergic system is expressed in human cytotrophoblast cells and in immune cells with homeostatic regulatory functions. AIM: The aim of this work was to evaluate whether non-neuronal acetylcholine conditions maternal monocyte and DC migration and activation profiles. METHODS: We used an in vitro model resembling maternal-placental interface represented by the co-culture of human trophoblast cells (Swan-71 cell line) and monocytes or DC. RESULTS: When cytotrophoblast cells were treated with neostigmine (Neo) to concentrate endogenous acetylcholine levels, monocyte migration was increased. In parallel, high levels of IL-10 and decreased levels of TNF-α were observed upon interaction of maternal monocytes with trophoblast cells. This effect was synergized by Neo and was prevented by atropine, a muscarinic acetylcholine receptor antagonist. Similarly, trophoblast cells increased the migration of DC independently of Neo treatment; however, enhanced IL-10 and MCP-1 synthesis in trophoblast-DC co-cultures with no changes in TNF-α and IL-6 was observed. In fact, there were no changes in HLA-DR, CD86 or CD83 expression. Finally, trophoblast cells treated with Neo increased the expression of two antigen-presenting cells attracting chemokines, MCP-1, MIP-1α and RANTES through muscarinic receptors, and it was prevented by atropine. CONCLUSIONS: Our present results support a novel role of acetylcholine synthesized by trophoblast cells to modulate antigen-presenting cell migration and activation favouring an immunosuppressant profile that contributes to immune homeostasis maintenance at the maternal-foetal interface.

Monocytes from Sjögren's syndrome patients display increased vasoactive intestinal peptide receptor 2 expression and impaired apoptotic cell phagocytosis.

Sjögren's syndrome (SS) is a chronic autoimmune disease characterized by salivary and lacrimal gland dysfunction. Clinical observations and results from animal models of SS support the role of aberrant epithelial cell apoptosis and immune homeostasis loss in the glands as triggering factors for the autoimmune response. Vasoactive intestinal peptide (VIP) promotes potent anti-inflammatory effects in several inflammatory and autoimmune disease models, including the non-obese diabetic (NOD) mouse model of SS. With the knowledge that VIP modulates monocyte function through vasoactive intestinal peptide receptors (VPAC) and that immune homeostasis maintenance depends strongly upon a rapid and immunosuppressant apoptotic cell clearance by monocytes/macrophages, in this study we explored VPAC expression on monocytes from primary SS (pSS) patients and the ability of VIP to modulate apoptotic cell phagocytic function and cytokine profile. Monocytes isolated from individual pSS patients showed an increased expression of VPAC2 subtype of VIP receptors, absent in monocytes from control subjects, with no changes in VPAC1 expression. VPAC2 receptor expression could be induced further with lipopolysaccharide (LPS) in pSS monocytes and VIP inhibited the effect. Moreover, monocytes from pSS patients showed an impaired phagocytosis of apoptotic epithelial cells, as evidenced by reduced engulfment ability and the failure to promote an immunosuppressant cytokine profile. However, VIP neither modulated monocyte/macrophage phagocytic function nor did it reverse their inflammatory profile. We conclude that monocytes from pSS patients express high levels of VPAC2 and display a deficient clearance of apoptotic cells that is not modulated by VIP.

Defects in the vasoactive intestinal peptide (VIP)/VPAC system during early stages of the placental-maternal leucocyte interaction impair the maternal tolerogenic response.

Successful embryo implantation occurs followed by a local inflammatory/T helper type 1 (Th1) response, subsequently redirected towards a tolerogenic predominant profile. The lack of control of this initial local inflammatory response may be an underlying cause of early pregnancy complications as recurrent spontaneous abortions (RSA). Considering that vasoactive intestinal peptide (VIP) mediates anti-inflammatory and tolerogenic effects in several conditions we hypothesized that VIP might contribute to tolerance towards trophoblast antigens during the early interaction of maternal leucocytes and trophoblast cells. In this study we investigated VIP/VPAC system activity and expression on maternal peripheral blood mononuclear cells (PBMCs) after interaction with immortalized trophoblast cells (Swan-71 cell line) as an in-vitro model of feto-maternal interaction, and we analysed whether it modulates maternal regulatory T cell (T(reg))/Th1 responses. We also investigated the contribution of the endogenous VIP/VPAC system to RSA pathogenesis. VIP decreased T-bet expression significantly, reduced monocyte chemotactic protein-1 (MCP-1) and nitrite production in co-cultures of PBMCs from fertile women with trophoblast cells; while it increased the frequency of CD4(+) CD25(+) forkhead box protein 3 (Foxp3)(+) cells, transforming growth factor (TGF)-ß expression and interleukin (IL)-10 secretion. These effects were prevented by VIP-specific antagonist. Interestingly, PBMCs from RSA patients displayed significantly higher T-bet expression, lower T(reg) frequency and lower frequency of VIP-producer CD4 lymphocytes after the interaction with trophoblast cells. Moreover, the patients displayed a significantly lower frequency of endometrial CD4(+) VIP(+) cells in comparison with fertile women. VIP showed a Th1-limiting and T(reg) -promoting response in vitro that would favour early pregnancy outcome. Because RSA patients displayed defects in the VIP/VPAC system, this neuropeptide could be a promising candidate for diagnostic biomarker or surrogate biomarker for recurrent spontaneous abortions.

Vasoactive intestinal peptide/vasoactive intestinal peptide receptor relative expression in salivary glands as one endogenous modulator of acinar cell apoptosis in a murine model of Sjögren's syndrome.

Sjögren's syndrome (SS) is a chronic autoimmune disease characterized by a progressive oral and ocular dryness that correlates poorly with the autoimmune damage of the glands. It has been proposed that a loss of homeostatic equilibrium in the glands is partly responsible for salivary dysfunction with acinar cells involved actively in the pathogenesis of SS. The non-obese diabetic (NOD) mouse model of Sjögren's syndrome develops secretory dysfunction and early loss of glandular homeostatic mechanisms, with mild infiltration of the glands. Based on the vasodilator, prosecretory and trophic effects of the vasoactive intestinal peptide (VIP) on acini as well as its anti-inflammatory properties we hypothesized that the local expression of VIP/vasoactive intestinal peptide receptor (VPAC) system in salivary glands could have a role in acinar cell apoptosis and macrophage function thus influencing gland homeostasis. Here we show a progressive decline of VIP expression in submandibular glands of NOD mice with no changes in VPAC receptor expression compared with normal mice. The deep loss of endogenous VIP was associated with a loss of acinar cells through apoptotic mechanisms that could be induced further by tumour necrosis factor (TNF)-α and reversed by VIP through a cyclic adenosine-5'-monophosphate (cAMP)/protein kinase A (PKA)-mediated pathway. The clearance of apoptotic acinar cells by macrophages was impaired for NOD macrophages but a shift from inflammatory to regulatory phenotype was induced in macrophages during phagocytosis of apoptotic acinar cells. These results support that the decline in endogenous VIP/VPAC local levels might influence the survival/apoptosis intracellular set point in NOD acinar cells and their clearance, thus contributing to gland homeostasis loss.

Expression of SLAM as a functional and phenotypic marker in women with recurrent miscarriage.

In the present work, we investigated the Th1 and Th2 cytokine patterns secreted by infiltrating endometrial lymphocytes from fertile women and from patients with recurrent spontaneous miscarriage (RSM). Moreover, we also analyzed the expression of cytokines in the whole endometrium from fertile and RSM women. Furthermore, we investigated the expression of the activation marker signaling lymphocytic activation molecule (SLAM) a cell surface glycoprotein expressed on lymphocytes that upon engagement boosts IFN-gamma production. Our results showed a slight increase in IL-10 expression in the endometrium of some fertile women, although no significant differences were found in IFN-gamma and IL-5 expression. In contrast, analysis of IFN-gamma production by polyclonal activated lymphocytes from endometrium and/or peripheral blood from fertile women showed a significant increase compared to RSM. Analysis of SLAM protein expression in luteal phase endometrial samples showed a significant increase in the levels of the receptor in RSM women compared to fertile women. These results correlated with a significant augmentation of SLAM levels in peripheral blood T-lymphocytes from RSM patients. Interestingly, after treatment of RSM patients with paternal mononuclear cells, surface-SLAM-expression in T-cells from RSM patients significantly decreased up to levels comparable to those of fertile women. Taken together, our results suggest that endometrial cells have not a defined pattern-cytokine-production under pre-implatatory conditions, and SLAM might be a potential marker for the diagnosis of RSM and an indicator useful to follow up the patient response to allogeneic immunotherapy.

Intracellular expression of CD69 in endometrial and peripheral T cells represents a useful marker in women with recurrent miscarriage: modulation after allogeneic leukocyte immunotherapy.

PROBLEM: To characterize in fertile women and women with recurrent spontaneous abortions (RSA) the expression and functional status of T cells expressing the CD69 molecule. METHOD OF STUDY: We analyzed by flow cytometry in peripheral blood and endometrium from fertile and RSA women, the surface and cytoplasmic expression of CD69 on gated T cells. In addition, we investigated by three-color flow cytometry the expression of cytokines, and subsets of memory T cells. RESULTS: In T cells, CD69 was restricted to the intracellular compartment with a higher frequency in RSA than in fertile women (68.2 +/- 12% versus 23.7 +/- 22%, P < 0.001, and 20 +/- 9.5% versus 2.1 +/- 3.8%, P < 0.005, in endometrium and peripheral blood, respectively). In contrast, the number of interferon-gamma+ (IFN-gamma+) secreting cells was higher (16 +/- 5% versus 6 +/- 1%) in fertile women. All 11 RSA women alloimmunized with parental leukocytes reached values of CD3 +/- CD69+ cells similar to those observed in fertile women. CONCLUSIONS: CD69 might represent a useful marker in the diagnosis and the follow up of RSA patients.

Induction of allogenic T-cell hyporesponsiveness by galectin-1-mediated apoptotic and non-apoptotic mechanisms.

Galectin-1, a beta-galactoside-binding protein expressed at sites of T-cell activation and immune privilege, has shown specific immunosuppressive properties. Because of the implications of this protein in T-cell tolerance and its potential use to avoid graft rejection, we investigated the immunosuppressive effects of galectin-1 in the course of the human allogenic T-cell response. Galectin-1 induced a dose- and carbohydrate-dependent inhibition of the allogenic T-cell response. Addition of galectin-1 to alloreactive lymphocytes resulted in significant apoptosis of CD45R0-positive cells. This negative regulatory effect was accompanied by caspase activation, Bcl-2 downregulation and was prevented by addition of exogenous IL-2. In addition, a significant decrease of IFN-gamma production was detected in the non-apoptotic cell population, following exposure of alloreactive lymphocytes to galectin-1. Moreover, the immunosuppressive activity of this protein did not involve TGF-beta-mediated mechanisms. Since galectin-1 is expressed by activated T cells and could be acting by an autocrine negative loop to control human T-cell reactivity, we finally examined the regulated expression of this protein throughout the allogenic T-cell response. Expression of endogenous galectin-1 was detected at 24 h of cell culture, reaching its maximal levels after 72 h of allostimulation. The present study sets the basis for a potential use of galectin-1 as a selective immunosuppressive agent to limit T-cell-mediated reactivity during the effector phase of the alloimmune response.

Modulation of CD44 in acute lymphoblastic leukemia identifies functional and phenotypic differences of human B cell precursors.

CD44 expression and other B cell markers were analyzed in 38 samples of B cell precursors (BCP) from patients with acute lymphoblastic leukemia (ALL). According to the expression of CD10 and CD44, we established the following five stages of BCP-ALL phenotypes that may represent different forms of interaction between BCP-ALL and bone marrow-adherent cells: stage 1, CD19+, CD44bright, CD10-; stage 2, CD19+, CD44bright, CD10dim/bright; stage 3, CD19+, CD44dim, CD10bright, CD20-/+; stage 4, CD19+, CD44dim, CD10dim, CD20+; and stage 5, CD19+, CD44bright, CD10-, CD20+. Next, we analyzed the modulation of CD44 according to the expression of the different BCP-ALL phenotypes by incubating the samples under different culture conditions, including addition of stromal cells and interleukin (IL)-7. In culture, the samples in stages 1 and 2 maintained high expression of CD44 and re-expressed this molecule when cultured after trypsin treatment, indicating ongoing synthesis of CD44. Similarly, the stage 3 samples cultured in the presence of stromal cells, IL-7, or both also upregulated CD44 expression in culture. In contrast, the low expression of CD44 on the presumably more mature stage 4 samples was not modified by the addition of stromal cells or IL-7 or when cultured after trypsin treatment, suggesting that those cells had arrested CD44 synthesis. We concluded that down-modulation of CD44 occurred in association with differentiation to phenotype stages 3 and 4 and we hypothesized that this down-modulation might be associated with the exit of BCP-ALL from the bone marrow.

Is the paternal mononuclear cells' immunization a successful treatment for recurrent spontaneous abortion?

PROBLEM: Alloimmunization as a treatment for recurrent spontaneous abortion (RSA) is still controversial due to the lack of enough controls to evaluate its effectiveness. The present study was conducted to compare the live birth rate in the presence or absence of immunotherapy. METHOD OF STUDY: Ninety-two women with RSA (79 primary [PA] and 13 secondary aborters[SA]) received immunotherapy. Thirty-seven RSA couples not receiving paternal alloimmunization, constituted the "control" group. RESULTS: The pregnancy rate in alloimmunized was 58 vs 46% in the control group. The live birth increased from 71% in the controls to 88% after immunotherapy. The alloimmunization induced mixed lymphocyte reaction blocking factors (MLR BFs) in 79% of women. However, they were also present in 83% of immunized women experiencing a new abortion. CONCLUSION: These results indicate that alloimmunization may be useful in the treatment of RSA.